CN109486784B

CN109486784B - Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered ring key intermediate

Info

Publication number: CN109486784B
Application number: CN201811449438.2A
Authority: CN
Inventors: 廖祥儒; 翟李欣; 赖英杰; 殷方荣; 蔡宇杰; 管政兵
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-06-09
Anticipated expiration: 2038-11-30
Also published as: CN109486784A

Abstract

The invention discloses a omega-transaminase mutant capable of catalyzing a sitafloxacin five-membered ring key intermediate, and belongs to the technical field of genetic engineering and enzyme engineering. The invention obtains the omega-transaminase mutant capable of catalyzing the sitafloxacin five-membered ring key intermediate by performing site-specific mutagenesis on amino acid with higher factor B in the crystal structure of the omega-transaminase, namely mutating Y at the position of the omega-transaminase to L, S at the position of 190 to A, L at the position of 212 to M and I at the position of 215 to M. The mutant Y32L/S190A/L212M/I215M of the invention has the catalytic efficiency Kcat/Km of 0.64min^‑1·mM^‑1Catalyzing (S) -5-benzyl-5-azaspiro [2.4]Generation of 5-benzyl-5-azaspiro [2.4] by heptan-7-amine]5-benzyl-5-azaspiro [2.4] mutant of heptan-7-one]The maximum conversion of heptan-7-one was 79.02%. The mutant obtained by the invention is suitable for the application of industrially and manually synthesizing the sitafloxacin five-membered ring key intermediate.

Description

Omega-transaminase mutant capable of catalyzing sitafloxacin five-membered ring key intermediate

Technical Field

The invention relates to a omega-transaminase mutant capable of catalyzing a sitafloxacin five-membered ring key intermediate, belonging to the technical field of genetic engineering and enzyme engineering.

Background

Sitafloxacin (Sitafloxacin Hydrate), a broad-spectrum quinolone antibacterial agent developed by the first pharmaceutical three-co-corporation of japan in 2008, has a good bactericidal effect on many clinically common fluoroquinolone-resistant strains, and is used for treating serious intractable infectious diseases. Compared with other quinolone medicines, the product has low daily treatment cost and good treatment effect and safety. But the key technical difficulty in the synthesis process is the asymmetric synthesis of the sitafloxacin five-membered ring key intermediate. The unstable, high-cost and difficult-to-scale technical limitation of the process severely limits the production of sitafloxacin. Compared with the chemical synthesis method of chiral amine drugs, the enzyme asymmetric catalysis of the biosynthesis method is concerned by the advantages of strong substrate specificity and stereoselectivity, mild reaction conditions, environmental friendliness and the like. Therefore, the enzyme asymmetric catalysis biosynthesis method has higher research and application values.

Transaminase (transaminase) belongs to transferase and is generally used for catalyzing amino group to be transferred from an amino donor compound to an amino acceptor compound, protein sequences of transaminase from different sources reported in the literature are compared and clustered according to differences of different variable regions, and then the transaminase is divided into 4 types according to superposition and iterative comparison of hydrophilic sites, wherein omega-transaminase belongs to a second subfamily and is generally used for preparing chiral amine and unnatural amino acid, such as β -amino acid.

In protein molecular engineering, methods commonly used at present are rational design, irrational design and semi-rational design. The main differences between the three methods are whether the molecular structure of the enzyme protein is well understood and whether calculations and predictions need to be made using bioinformatics software. The rational design has the advantages of low experimental cost, simplicity, convenience, short time and the like.

Although the omega-transaminase has great application and research values, the omega-transaminase screened from wild bacteria cannot specifically catalyze sitafloxacin five-membered ring key intermediates, so that the omega-transaminase cannot be further applied to industrial production, and the development and application are greatly reduced.

Disclosure of Invention

In order to solve the problems, the invention carries out heterologous expression and site-directed mutation modification on omega-transaminase (the omega-transaminase from Bacillus pumilus and the application of the omega-transaminase in biological amination. 201811219769.7) from Bacillus pumilus W3, so that the modified omega-transaminase can specifically catalyze sitafloxacin five-membered ring key intermediates, and has profound technical guidance significance for industrial application and popularization of the omega-transaminase.

Compared with parent omega-transaminase, the omega-transaminase mutant can specifically catalyze sitafloxacin five-membered ring key intermediate. The parent gene is consistent with a Bacillus pumilus (Bacillus pumilus W3) omega-transaminase gene (Sequence ID: MH196528), and the plasmid template used for mutation is a carrier ota3/pCold II (application number: CN201811219769.7) carrying a natural omega-transaminase coding gene.

The invention aims to provide a omega-transaminase mutant capable of specifically catalyzing sitafloxacin five-membered ring key intermediate, and the amino acid sequence of the mutant comprises the following components: 1, the amino acid sequence obtained by simultaneously mutating tyrosine at the 32 nd position to leucine, serine at the 190 th position to alanine, leucine at the 212 th position to methionine, and isoleucine at the 215 th position to methionine on the basis of the amino acid sequence of SEQ ID NO. 1 is named as Y32L/S190A/L212M/I215M.

In one embodiment, the amino acid sequence of the ω -transaminase mutant is the sequence shown in SEQ ID NO. 5.

In one embodiment, the nucleotide sequence of the ω -transaminase mutant comprises the sequence shown in SEQ ID NO. 2.

A second object of the present invention is a method for preparing said mutant, comprising the steps of:

(1) designing primers for site-directed mutagenesis, carrying out mutagenesis by taking a vector carrying a omega-transaminase coding gene as a template, and constructing a plasmid vector of the Y32L/S190A/L212M/I215M mutant;

(2) and (3) transforming the recombinant plasmid with the correct sequence into escherichia coli BL21(DE3) to obtain a recombinant bacterium, fermenting and culturing the recombinant bacterium, and obtaining fermentation supernatant fluid containing the omega-transaminase mutant.

In one embodiment, the fermentation is by culturing the recombinant bacteria to OD at 37 ℃₆₀₀After that, the temperature was decreased to 15 ℃ and IPTG was added to a final concentration of 0.4mM for induction, and the mixture was centrifuged to obtain a supernatant enzyme solution after 24 hours of culture.

In one embodiment, the preparation method further comprises purifying the ω -transaminase in the fermentation supernatant using an AKTA protein purifier and a histrappf fraction 1ml nickel column.

The third purpose of the invention is to provide a recombinant plasmid vector containing the amino acid sequence of the mutant.

In one embodiment, the plasmid vector is any one of pET series, pGEX series, pCold series, or pUB.

The invention also claims a gene for coding the mutant and a gene engineering bacterium for expressing the mutant.

The invention also claims the application of the mutant, the gene for coding the mutant and the gene engineering bacteria for expressing the mutant in the catalytic synthesis of related chiral amine in the aspects of food, chemical engineering or medicament preparation, in particular the application in the preparation of sitafloxacin medicaments.

In one embodiment, the use comprises catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound.

The applications include the selective catalysis and chiral synthesis of chiral amines, such as (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine and other unnatural amino acids.

The invention has the beneficial effects that:

the omega-transaminase mutant is mutated on the basis of R type omega-transaminase derived from Bacillus pumilus, and the constructed omega-transaminase mutant Y32L/S190A/L212M/I215M has the performance of specifically catalyzing sitafloxacin five-membered ring key intermediates. The enzyme kinetic analysis shows that the K of the mutant Y32L/S190A/L212M/I215M of the invention_mA value of 4.88. + -. 0.20 mM; catalytic efficiency K_cat/K_mIs 0.64min^-1mM^-1. Catalysis of (S) -5-benzyl-5-azaspiro [2.4]]Generation of 5-benzyl-5-azaspiro [2.4] by heptan-7-amine]Heptan-7-one, 5-benzyl-5-azaspiro [2.4] of the mutant of the present invention]The maximum conversion of heptan-7-one was 79.02%. The omega-transaminase parent cannot catalyze the sitafloxacin five-membered ring key intermediate (S) -5-benzyl-5-azaspiro [2.4]]heptan-7-amine. Therefore, the omega-transaminase mutant is more suitable for catalyzing (S) -5-benzyl-5-azaspiro [2.4] by the omega-transaminase than the parent]Application in chiral amine process such as heptan-7-amine.

Drawings

FIG. 1: a three-dimensional simulation structure of natural omega-transaminase;

FIG. 2: chemical structure of key intermediate of Sitafloxacin five-membered ring;

FIG. 3: performing SDS-PAGE gel electrophoresis on the natural omega-transaminase and the mutant pure enzyme; wherein, lane 1 represents the protein molecular weight standard, lane 2 is mutant Y32L/S190A/L212M/I215M, and lane 3 is natural ω -transaminase.

Detailed Description

Example 1: preparation and construction of omega-transaminase site-directed mutants

Omega-transaminase 1 site-directed mutant from Bacillus pumilus W3Y 32L/S190A/L212M/I215M:

in the invention, a three-dimensional simulation structure of Bacillus pumilus W3 omega-transaminase (omega-BPAT) is constructed by a Swiss-Model online server by taking a crystal structure (PDB ID:5E25) of the thermophilic archaea transaminase with the highest similarity as a template (FIG. 1). Through amino acid primary sequence alignment, the similarity between the thermophilic archaea transaminase and the omega-BPAT reaches 51.21 percent, and accords with the parameters of homology modeling, so that the omega-BPAT can be considered to have a three-dimensional structure similar to the thermophilic archaea transaminase. Based on the results predicted by software analysis, mutant Y32L/S190A/L212M/I215M was constructed using PCR-mediated site-directed mutagenesis.

The preparation method of the site-directed mutant comprises the steps of respectively designing and synthesizing primers for introducing site-directed mutation according to the sequence (the amino acid sequence is shown as SEQ ID NO: 1) of Bacillus pumilus W3 omega-transaminase, simultaneously carrying out site-directed mutation on the positions of omega-transaminase Y32, S190, L212 and I215, determining a DNA coding sequence, and respectively sequencing to confirm whether the coding gene of the omega-transaminase mutant is correct; the mutant gene is connected to a proper expression vector (any one of pET series, pGEX series, pCold series or pUB) and is introduced into escherichia coli for expression, and the corresponding omega-transaminase site-directed mutant is obtained.

PCR amplification of site-directed mutant coding gene: using PCR technology, expression vector ota3/pCold II was used as template.

The mutation primers for introducing the site-directed mutation of Y32L/S190A/L212M/I215M are as follows:

BPTA-F1：5’-TTGGCGCAATGGAGGGTATGACCCGCAACGCAATTA-3’(SEQ ID NO:3)

BPTA-R1：5’-CGGGTCATACCCTCCATTGCGCCAATATAACCTGGC-3’(SEQ ID NO:4)

BPTA-F2：5’-GTAGCCGAAGGGGCGGCTGATAATGTTTTTATCTAT-3’(SEQ ID NO:6)

BPTA-R2：5’-CATTATCAGCCGCCCCTTCGGCTACATATCCTTGAT-3’(SEQ ID NO:7)

BPTA-F3：5’-CATGGGTTCTTACTTGGCGATGGGGTCTTCGAAGGC-3’(SEQ ID NO:8)

BPTA-R3：5’-CCCCATCGCCAAGTAAGAACCCATGGTCGTATACAG-3’(SEQ ID NO:9)

the PCR amplification program was set up as follows: first, pre-denaturation at 95 ℃ for 3 min; then 25 cycles were entered: denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 1min for 40 s; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel.

And (3) after purifying the PCR product, adding DPn I, heating at 37 ℃ in a water bath for 1h, degrading a template, assembling the mutant fragment, transforming E.coli JM109, selecting a positive clone, culturing in an LB liquid culture medium for 8-10h, preserving a glycerol tube, and sequencing. The mutant with correct sequencing (the amino acid sequence is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO: 2) is inoculated to an LB culture medium from a glycerol tube, overnight culture is carried out, plasmids are extracted, and the plasmids are transformed to express host escherichia coli BL21(DE3) competent cells, so as to obtain the recombinant strain capable of expressing the mutant Y32L/S190A/L212M/I215M.

Example 2: expression and purification of natural omega-aminotransferase and site-directed mutants thereof

Selecting a positive monoclonal transferred into an expression host escherichia coli BL21(DE3), growing for 8-10h in an LB liquid culture medium (containing 30 mug/mL ampicillin), and inoculating the seed fermentation liquid to the LB liquid culture medium (containing 30 mug/mL ampicillin) according to the inoculation amount of 5%; culturing Escherichia coli at 37 deg.C for 2 hr to OD₆₀₀About 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.05mM into the recombinant strain Y32L/S190A/L212M/I215M to induce extracellular expression, continuously culturing and fermenting for 24h at 15 ℃ in a shaking table, centrifuging the fermentation liquor at 4 ℃ and 8000g for 10min to remove thalli, and collecting the supernatant of centrifugal fermentation. Slowly adding 60% (NH) into the mutant fermentation supernatant under low-speed stirring by a magnetic stirrer₄)₂SO₄Salting out was performed overnight at 4 ℃. Centrifuging at 4 deg.C and 10000g for 20min, and collecting precipitate. After re-dissolving the precipitate with 50mmol/L of citric acid-disodium hydrogen phosphate buffer solution at pH 5.3, the precipitate was dialyzed overnight against 50mmol/L of citric acid-disodium hydrogen phosphate buffer solution at pH 5.3, while changing the dialysis buffer solution 2 to 3 times, and a sample was prepared by filtration through a 0.22 μm membrane. And (3) purifying the recombinant protein by adopting an AKTA avant protein purifier, wherein the temperature in the whole purification process is controlled to be 4 ℃. Cation exchange chromatography purification step: (1) balancing: equilibrating the strong cation exchange chromatography column with 5 volumes of 50mmol/L pH 5.3 citrate-disodium phosphate buffer; (2) loading: sampling the pretreated sample at the flow rate of 1 mL/min; (3) and (3) elution: comprises eluting unadsorbed substances, heteroproteins and target proteins at a flow rate of 1.0mL/min, wherein the eluent is 50% containing 1M NaClPerforming linear elution with citric acid-disodium hydrogen phosphate buffer solution of mmol/L pH 5.3, detecting wavelength of 280nm, and collecting eluate containing sucrose isomerase; only one target protein elution peak appears in the elution process, and enzyme activity is detected subsequently and SDS-PAGE protein electrophoresis shows that the enzyme solution collected at the peak top is the purest part no matter whether the enzyme solution is a wild type or a mutant. As shown in fig. 3.

Example 3: enzyme activity analysis method

The method for measuring the activity of ω -transaminase is described in Gao, S. (Gao, S., Su, y., Zhao, l., Li, g., Zheng, g.,2017. mutation of a (R) -selective amine transferase from fusarium oxygen system. process. biochem.63, 130-136.).

An appropriate amount of cell supernatant (or purified diluted enzyme solution) was added to 500. mu.L of sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing 20mM (R) - α -phenylethyylamine (or (S) - α -phenylethyylamine), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (PLP), and the mixture was mixed, reacted at 45 ℃ for 15 minutes, and then the reaction was terminated by adding an equal amount of ethyl acetate, and the absorbance of the solution at 254nm was measured before and after the reaction.

The amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute under the above conditions is defined as one enzyme activity unit (U/ml). The process is illustrated by △ A₂₅₄Calculating the enzyme activity of omega-transaminase, U/ml (△ A/min) V/rvb, △ A/min-absorbance change, V-reaction system volume (ml), r-molar extinction coefficient (cm)²/umol); v-sample size (ml); b-cuvette optical path length (cm), the above amounts can be increased or decreased proportionally.

The crude enzyme activity of the natural enzyme on the substrate (R) - α -phenylethynylamine is 1.1760U/mL, but the enzyme activity on (S) - α -phenylethynylamine is not good, and the recombinant mutant omega-aminotransferase of the invention has no activity on (R) - α -phenylethynylamine (or (S) - α -phenylethynylamine).

Example 4: application of mutant in production of sitafloxacin five-membered ring key intermediate (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine

Omega-transaminases can be divided into R-omega-transaminases and S-omega-transaminases, and different types of omega-transaminases can catalyze different types of substrates and thus can produce products with different optical properties. In the embodiment, the (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine (or (R) -5-benzyl-5-azaspiro [2.4] heptan-7-amine) is used as an amino donor, sodium pyruvate is used as an amino acceptor, and the catalytic capability of the obtained recombinase and the mutant recombinase on the amino donor is detected, so that whether the recombinase can specifically and efficiently catalyze the (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine is judged, and the application of the recombinase in the industrial synthesis of the sitafloxacin five-membered ring key intermediate is determined.

Chiral synthesis catalysis experiment: an appropriate amount of the purified diluted enzyme solution was added to 500. mu.L of sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing 20mM of (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine (or (R) -5-benzyl-5-azaspiro [2.4] heptan-7-amine), 20mM of sodium pyruvate, and 0.1mM of pyridoxal 5' -phosphate (PLP), mixed, reacted at 45 ℃ for 15min, respectively, and then the reaction was terminated by adding an equal amount of ethyl acetate. Centrifuging at 12,000 Xg for 1min, collecting the upper organic phase, filtering with 0.22 μm filter membrane, and detecting by High Performance Liquid Chromatography (HPLC) to obtain 5-benzyl-5-azaspiro [2.4] heptan-7-one.

Detection conditions are as follows:

column: agilent C18 column (250 × 4.6mm, Agilent, USA); mobile phase: acetonitrile/water (95/5, v/v); flow rate: 1 mL/min; detection wavelength: 220 nm.

TABLE 1

And (3) comparison: and (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine is used as a substrate, and the catalytic ability of the natural omega-transaminase to the amino acid is detected under the reaction conditions.

Test samples: the catalytic ability of the omega-transaminase Y32L/S190A/L212M/I215M mutant on (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine serving as a substrate is detected under the reaction conditions.

The data show that the catalytic activity of the recombinase on an experimental group (taking the omega-transaminase Y32L/S190A/L212M/I215M mutant as a catalyst) is better than that of a control group (taking the natural omega-transaminase as a catalyst). The data show that the recombinant omega-transaminase Y32L/S190A/L212M/I215M mutant has the function of efficiently and selectively synthesizing sitafloxacin five-membered ring key intermediates, and has large application potential (see Table 2).

TABLE 2

Enzyme activity (U/mg): the amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute is defined as one unit of enzyme activity (U/mg).

Example 5: determination of kinetic parameters of omega-transaminase mutants

Kinetic parameters of ω -transaminase were determined by reference to Gao, S. (Gao, S., Su, y., Zhao, l., Li, g., Zheng, g.,2017. characteristics of a (R) -selective amine transferase from fusarium oxysporum. process. biochem.63, 130-136.).

This example measured the kinetic parameters of the enzyme mutant Y32L/S190A/L212M/I215M (amino acid sequence shown in SEQ ID NO: 5) purified in example 2 at 45 ℃. The specific implementation method comprises the following steps: an appropriate amount of the cell supernatant (or the diluted purified enzyme solution) was collected, and 500. mu.L of a sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine (for example, concentration gradient see the above-mentioned publication), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (PLP) were added thereto, mixed, reacted at 45 ℃ for 15min, and then the reaction was stopped by adding an equal amount of ethyl acetate. The results of the kinetic studies are shown in table 3.

The results show that K of mutant Y32L/S190A/L212M/I215M_mThe value was 4.88 mM. Furthermore, the catalytic constant K of Y32L/S190A/L212M/I215M_catIs 3.1min^-1. Catalytic efficiency K of mutant Y32L/S190A/L212M/I215M_cat/K_mIs 0.64min^-1·mM^-1。

As shown in FIG. 1, Y32L/S190A/L212M/I215M is located in the catalytic center and the isomerization region of omega-transaminase, and mutation may cause the structure of the catalytic center and other partial regions of the isomerization region to be enlarged, so that larger substrates can enter the catalytic center, thereby possibly having positive influence on the substrate specificity and kinetic parameters of the enzyme.

TABLE 3 kinetic parameters of the ω -transaminase mutants

The conversion rate is the conversion efficiency of (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine into 5-benzyl-5-azaspiro [2.4] heptan-7-one.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. An omega-transaminase mutant is characterized in that the amino acid sequence of the omega-transaminase mutant is the sequence shown in SEQ ID NO. 5.

2. A gene encoding the mutant of claim 1.

3. A recombinant plasmid vector containing the nucleotide sequence of the gene of claim 2.

4. The recombinant plasmid vector according to claim 3, wherein the recombinant plasmid vector is constructed on the basis of any one of the plasmid vectors of pET series, pGEX series, pCold series, or pUB series.

5. A genetically engineered bacterium expressing the mutant of claim 1.

6. The mutant of claim 1, or the gene coding for the mutant of claim 1, or the genetically engineered bacterium expressing the mutant of claim 1, in the fields of food, chemical industry or medicine preparation, wherein the application is the synthesis of sitafloxacin five-membered ring key intermediate by catalyzing (S) -5-benzyl-5-azaspiro [2.4] heptan-7-amine.